The invention concerns biocompatible polymer systems comprising at least one polymer with a plurality of pores, said polymer comprising a sulfonic acid salt functionality designed to adsorb a broad range of protein based toxins from less than 0.5 kDa to 1,000 kDa and positively charged ions including but not limited to potassium.
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24. A biocompatible polymer system comprising at least one polymer, said polymer comprising (i) a plurality of pores and (ii) a sulfonic acid salt functionality;
said polymer system capable of adsorbing protein based toxins, inflammatory mediators and positively charged ions;
wherein the polymer comprises residues of one or more monomers selected from acrylonitrile, butyl acrylate, butyl methacrylate, cetyl acrylate, cetyl methacrylate, divinylbenzene, ethyl acrylate, ethyl methacrylate, ethylstyrene, methyl acrylate, methyl methacrylate, octyl acrylate, octyl methacrylate, styrene, vinylbenzyl alcohol, vinylformamide, vinylnaphthalene, and vinyltoluene;
wherein the polymer's pore structure has a total volume of pore sizes in the range of from 10 Å to 40,000 Å greater than 0.1 cc/g and less than 5.0 cc/g dry polymer; and
the biocompatible polymer system has improved biocompatibility by use of (i) a coating selected from a group consisting of poly(hydroxyethyl methacrylate), poly(hydroxyethyl acrylate), poly(dimethylaminoethyl methacrylate), salts of poly(acrylic acid), salts of poly(methacrylic acid), poly(diethylaminoethyl methacrylate), poly(hydroxypropyl methacrylate), poly(hydroxypropyl acrylate), poly(N-vinylpyrrolidone), poly(vinyl alcohol), heparin-mimicking polymers and mixtures thereof or (ii) a coating comprising heparin; and
wherein the polymer is in the form of a macroreticular porous polymer that has been sulfonated under mild conditions that retain residual functionality of any unreacted double bonds.
1. A biocompatible polymer system comprising at least one polymer, said polymer comprising (i) a plurality of pores and (ii) a sulfonic acid salt functionality;
said polymer system capable of adsorbing protein based toxins, inflammatory mediators and positively charged ions;
wherein the polymer comprises residues of one or more monomers selected from acrylonitrile, butyl acrylate, butyl methacrylate, cetyl acrylate, cetyl methacrylate, divinylbenzene, ethyl acrylate, ethyl methacrylate, ethylstyrene, methyl acrylate, methyl methacrylate, octyl acrylate, octyl methacrylate, styrene, vinylbenzyl alcohol, vinylformamide, vinylnaphthalene, and vinyltoluene;
wherein the polymer's pore structure has a total volume of pore sizes in the range of from 10 Å to 40,000 Å greater than 0.1 cc/g and less than 5.0 cc/g dry polymer; and
the biocompatible polymer system has improved biocompatibility by use of (i) a coating selected from a group consisting of poly(hydroxyethyl methacrylate), poly(hydroxyethyl acrylate), poly(dimethylaminoethyl methacrylate), salts of poly(acrylic acid), salts of poly(methacrylic acid), poly(diethylaminoethyl methacrylate), poly(hydroxypropyl methacrylate), poly(hydroxypropyl acrylate), poly(N-vinylpyrrolidone), poly(vinyl alcohol), heparin-mimicking polymers and mixtures thereof or (ii) a coating comprising heparin; and
wherein the polymer is in the form of a crosslinked polymer or a porous polymer; wherein the crosslinked polymer or porous polymer has been sulfonated under mild conditions that retain residual functionality of any unreacted double bonds and chloromethyl groups.
2. The biocompatible polymer system of
3. The biocompatible polymer system of
5. The biocompatible polymer system of
6. The biocompatible polymer system of
7. The biocompatibility imbuing modification of
8. The biocompatible polymer system of
9. The biocompatible polymer system of
10. The biocompatible polymer system of
12. The biocompatible polymer system of
14. The biocompatible polymer system of
15. The biocompatible polymer system of
16. The biocompatible polymer system of
17. The biocompatible polymer system of
18. The biocompatible polymer system of
19. The biocompatible polymer system of
20. The biocompatible polymer system of
21. The biocompatible polymer system of
22. The biocompatible polymer system of
23. The biocompatible polymer system of
25. A method of perfusion comprising passing a physiologic fluid once through or by way of an extracorporeal circuit through a device comprising the biocompatible polymer system of
26. A device for removing protein based toxins, inflammatory mediators and positively charged ions from physiologic fluid comprising the biocompatible polymer system of
27. The device of
28. The device of
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The present application claims priority to international patent application no. PCT/US2016/058019 which was filed Oct. 21, 2016 and to U.S. Provisional Patent Application No. 62/245,071 filed on Oct. 22, 2015. The contents of each that application are hereby incorporated by reference in their entirety.
The subject matter disclosed herein was made with government support under contract number HHSN268201600006C, awarded by The National Heart, Lung, and Blood Institute (NHLBI). The subject matter disclosed herein was also made with government support under contract number W81XWH-12-C-0038, awarded by The Department of Defense Small Business Innovation Research (DOD-SBIR). The government has certain rights in the herein disclosed subject matter.
The disclosed inventions are in the field of porous polymeric sorbents. The disclosed inventions are also in the field of broadly reducing contaminants in blood and blood products that can cause transfusion reactions; including, but not limited to, potassium, free hemoglobin, cytokines, bioactive lipids, and immunoglobulins. Additionally, the disclosed inventions are in the field of broadly removing contaminants by perfusion or hemoperfusion after tissue destruction; including, but not limited to, potassium, free hemoglobin, free myoglobin, cytokines, bioactive lipids, and immunoglobulins.
Packed red blood cell (pRBC) units contain reactive donor antibodies, free hemoglobin, high extracellular potassium levels, and biologically active inflammatory mediators that have the potential to cause adverse effects during blood transfusions. Such adverse effects can include non-hemolytic febrile and allergic transfusion reactions, atypical infections, allo-immunization, and potentially fatal reactions, like transfusion related acute lung injury (TRALI). Furthermore, transfusion risk increases in patients receiving multiple pRBCs, such as those involved in trauma or undergoing surgery, and in primed susceptible patients, such as those in critical care or undergoing high-risk surgery.
The likelihood of adverse effects increases over time for stored blood or blood products, as concentrations of many biological response modifiers, such as potassium, free hemoglobin, and cytokines, increase with storage duration. Cytokines are produced by residual leukocytes during storage of platelets and pRBCs, and can cause inflammation, fever, and direct vascular and organ injury. Erythrocytes contain phosphatidyl choline, and cytosolic and membrane phospholipase A2, contributing increasing levels of lysophosphatidylcholine (lysoPC) during storage. Structural and biochemical changes that RBCs undergo are described as “storage lesion” and lead to a progressive loss of hemoglobin, and potassium. Plasma free hemoglobin can rapidly overwhelm the scavenging capability of haptoglobin, resulting in oxidative damage to lipids, proteins, endothelial cells, tissues, and renal proximal tubules, and in depletion of nitric oxide (NO) upon transfusion. Increases in extracellular potassium during storage lead to an increased risk of hyperkalemia and arrhythmia, particularly for large volume or “massive” transfusions and transfusions in newborns and infants.
Hyperkalemia describes a condition in which the potassium level in the blood exceeds a concentration of 5 mEq/L, where concentrations exceeding 7 mEq/L are considered severe cases. The electrical rhythm of the heart can be altered by moderate hyperkalemia, while severe conditions may cause the heart to stop beating. In addition to blood transfusions, another major cause of hyperkalemia is tissue destruction that causes dying cells to release potassium into blood circulation. Tissue destruction typically results from trauma, burns, hemolysis, massive lysis of tumor cells, rhabdomyolysis, or major surgery, such as cardiac surgery or cardiopulmonary bypass (CPB), where severe tissue destruction leads to more severe cases of hyperkalemia. In addition to the release of potassium into blood circulation, massive tissue injury is characterized by release of a large amount of myoglobin from damaged muscle tissue, plasma free hemoglobin from hemolyzed red blood cells, damage associated molecular pattern (DAMP) factors from damaged cells, and an upregulation of pro- and anti-inflammatory mediators, such as cytokines. Excessive free myoglobin, free hemoglobin, and other inflammatory mediators, can lead to complications such as renal failure or even death. Abnormal regulation of cytokines, or release of DAMPS, may lead to systemic inflammatory response syndrome (SIRS) and multi-organ dysfunction (MODs).
Currently, there are existing technologies for potassium removal, or antibody removal, from stored blood or blood products. Kawasumi Laboratories has developed a single-pass in-line potassium adsorption filter to reduce the risk of hyperkalemia and improve safety for blood transfusions. The filter functions by exchanging potassium ions (K+) for sodium ions (Na+) to decrease the concentration of K+ in stored RBC units. In an in-vitro study conducted by Yamada et. al, 10 filters were tested using each of three AS-3 RBC units via gravity filtration. The mean decrease in potassium was 97.5%, 91.2%, and 64.4% for the first, second, and third units, respectively. Accompanying the decrease in potassium were mean increases of sodium by 33%, magnesium by 151.4%, and total calcium by 116.1%. Plasma hemoglobin was unchanged after filtration.
A journal article published by Terai et. al., titled “Development of a Potassium-Specific Adsorbent for Direct Hemoperfusion”, describes a study assessing the development of a sodium/calcium/magnesium exchange resin mixture that removes potassium without associated electrolyte abnormalities. At the time the article was written, direct hemoperfusion over an exchange resin was capable of lowering elevated serum potassium levels, but had not been used clinically due to subsequent electrolyte abnormalities. Prior to evaluating the exchange resin in an in vivo model, batch experiments were conducted in vitro to identify an effective ratio of sodium to calcium to magnesium for the resin mixture. Results from the study demonstrated a reduction of elevated plasma potassium levels from about 6.7 to about 3.5 mEq/L in anephric dogs, without any significant change in levels of sodium, calcium, magnesium, albumin, total protein, or cholesterol, after 2 hours of direct hemoperfusion through an exchange resin column. Pre- and post-hemoperfusion platelet counts and plasma free hemoglobin levels were also measured, where post-hemoperfusion platelet counts were only about 45% of pre-hemoperfusion levels, and there was no significant change in plasma free hemoglobin levels.
Patent WO 2012118735 A2, entitled “Removal of immunoglobulins and leukocytes from biological fluids,” discloses devices, systems, and methods, for depleting biological fluids of immunoglobulins and leukocytes. It describes a system comprising immunoglobulin binding media and a leukocyte depletion filter element, where the binding media consist of cellulose beads and are placed into the pre-filtration blood bag. In one example, 30 g dry weight cellulose beads, (4-MEP) HyperCer™ chromatography sorbent (Pall Corporation), were placed in a blood bag to which a unit of 5 day old AS-3 RBC was added, and the blood bag mixed on a rotamixer. The RBCs were gravity filtered through a downstream filter, where beads were trapped in an immunoglobulin binding media chamber and filtered cells passed through a fibrous leukocyte depletion filter before being collected and analyzed. Leukocyte content was reduced by 5.17 log, IgA reduced by 81%, IgG by 98%, and IgM by 42%. In another example, the ability of the leukocyte filter to remove cytokines was examined. Two units of 22-30 day old ABO compatible red cell concentrate were pooled together and then split into two lots. The first was placed in a blood bag containing about 25-33 g dry weight cellulose beads, (4-MEP) HyperCer™ chromatography sorbent (Pall Corporation), with 10 mL PBS and mixed for 45 minutes, and the second passed through a BPF4 High Efficiency leukocyte depletion filter (Pall Corporation) via gravity filtration. Afterwards, both lots were analyzed and it was found that in the aliquot placed in contact with the beads, interleukin 1-Beta (IL-1β) was reduced by 45.7%, interleukin-6 (IL-6) by 26.9%, interleukin-8 (IL-8) by 57.1% and tissue necrosis factor-alpha (TNF-α) by 49.9% For the aliquot passed through the filter, IL-1β was not reduced, IL-6 was not reduced, IL-8 was reduced by 35.0% and TNF-α reduced by 7.5%
In a journal article by Silliman et. al., it was demonstrated that pre-storage filtration of packed RBCs removes HLA and HNA antibodies, reducing pro-inflammatory activity in RBC supernatant in an animal TRALI model. In the described study, plasma that contained antibodies to human lymphocyte antigen (HLA)-A2, or human neutrophil antigen (HNA)-3a, was filtered and priming activities of specific HNA-3a and HLA-2a were measured. OX27 antibodies were added to plasma and filtration was analyzed using a 2-event animal model for TRALI. RBC units from 31 donors, who were known to possess antibodies against HLA antigens, were filtered. In addition, 4 RBC units underwent standard leukoreduction. PMN priming activity, immunoglobulins, HLA antibodies, and ability to induce TRALI were measured. Filtration of the plasma was shown to remove more than 96% of IgG, and antibodies to HLA-A2 and HNA-3a, including their respective priming activity, and mitigated in vivo TRALI. Antibodies to HLA antigens were removed in experimental filtration of RBC units, accompanied by an inhibition of accumulation of lipid priming activity and lipid-mediated TRALI.
The sorbent material described herein is uniquely designed to efficiently remove free hemoglobin, antibodies, bioactive lipids, cytokines, and potassium, from blood and blood products. The polymer is multi-functional, retaining said biomolecules through tortuous path, sorption, pore capture, and ion exchange mechanisms. Novel chemistry is used to synthesize the polymer, utilizing a controlled sulfonation procedure that allows for the incorporation of sulfonic acid groups onto the aromatic rings without oxidizing all residual double bonds. This allows the polymeric matrix to maintain protein sorption and ion exchange capabilities, while still leaving residual functional groups available for hemocompatibility improvement modifications. The balance between sulfonation and retention of residual double bonds is crucial for preparation of an effective polymer sorbent.
Differentiating the multi-functional polymer from other filters that remove only reactive proteins or only potassium is its ability to remove both simultaneously without sacrificing binding capacity for either. Additionally, the sorbent is able to remove cytokines and inflammatory protein moieties simultaneously while removing potassium and antibodies. For hemoperfusion applications, it is a requirement that the polymer is hemocompatible. Using the unactivated partial thromboplastin time (uPTT) assay as a measure of thrombogenicity, the polymer described herein exhibits minimal activation, indicating a plasma-like interaction. This polymer is suited for a wide variety of applications, as many cases of trauma, burn, and major surgery, result in hyperkalemia, cytokine storm, and require blood transfusions. The ability to use one multi-application filter has many advantages over using many single-application filters. Given the value of blood and blood products, the use of a single, smaller filter that minimizes cell loss within the retained volume and reduces complexity of material quality assurance is very desirable.
In some aspects, the invention concerns biocompatible polymer system comprising at least one polymer, said polymer comprising (i) a plurality of pores and (ii) a sulfonic acid salt functionality; the polymer system capable of adsorbing (i) a broad range of protein based toxins having a molecular weight of from less than about 0.5 kDa to about 1,000 kDa (or about 1 kDa to about 1,000 kDa in some embodiments) and (ii) positively charged ions. Some polymer systems have a polymer pore structure that has a total volume of pore sizes in the range of from 10 Å to 40,000 Å greater than 0.1 cc/g and less than 5.0 cc/g dry polymer. Some preferred polymers are hemocompatible. The polymer system has the form of a solid support. Certain preferred polymer systems have a geometry of a spherical bead. Other polymer systems have the form of a fiber, monolithic column, film, membrane, or semi-permeable membrane.
In some embodiments, the toxins adsorbed comprise one or more of inflammatory mediators and stimulators comprised of one or more of cytokines, superantigens, monokines, chemokines, interferons, proteases, enzymes, peptides including bradykinin, soluble CD40 ligand, bioactive lipids, oxidized lipids, cell-free hemoglobin, cell-free myoglobin, growth factors, glycoproteins, prions, toxins, bacterial and viral toxins, endotoxins, drugs, vasoactive substances, foreign antigens, antibodies, and positively charged ions. In some preferred embodiments, the positively charged ion is potassium.
The polymers can be made by any means known in the art to produce a suitable porous polymer. In some embodiments, the polymer is made using suspension polymerization. Some polymers comprise a hypercrosslinked polymer. Certain spherical beads have a biocompatible hydrogel coating. In certain embodiments, the polymer is in the form of hypercrosslinked or a macroreticular porous polymer beads that have been sulfonated under mild conditions that retain residual functionality of any unreacted double bonds and chloromethyl groups. The unreacted double bonds or chloromethyl groups can be modified via free radical or SN2 type chemistry to attach one or more of biocompatible and hemocompatible monomers, cross-linkers or low molecular weight oligomers.
In some embodiments, the porous polymer beads comprise sulfonic acid groups or a salt thereof, sulfonyl chloride, or sulfonate ester groups. The polymer beads comprising sulfonic acid groups or a salt thereof, sulfonyl chloride, or sulfonate ester groups can be produced by graft copolymerization of (i) premade porous polymer that contains unreacted double bonds with (ii) polymerizable vinyl monomers containing sulfonic acid groups or a salt thereof to form a mixture comprising hemocompatible vinyl monomers.
Some polymer systems are constructed from polymerizable vinyl monomers containing sulfonic acid groups or a salt thereof which are copolymerized in the presence of cross-linker, hemocompatible monomer, monomer and suitable porogen to yield porous polymeric polymer containing a sulfonic acid salt functionality.
Certain polymers are formed and subsequently modified to be biocompatible. Some modifications comprise forming a biocompatible surface coating or layer.
Other aspects include methods of perfusion comprising passing a physiologic fluid once through or by way of a suitable extracorporeal circuit through a device comprising the biocompatible polymer system described herein.
Yet another aspect concerns devices for removing (i) a broad range of protein based toxins from less than 0.5 kDa to 1,000 kDa and (ii) positively charged ions from physiologic fluid comprising the biocompatible polymer system described herein.
The accompanying drawings, which are included to provide a further understanding of the disclosure, are incorporated in and constitute a part of this specification, illustrate aspects of the disclosure and together with the detailed description serve to explain the principles of the disclosure. No attempt is made to show structural details of the disclosure in more detail than may be necessary for a fundamental understanding of the disclosure and the various ways in which it may be practiced. In the drawings:
As required, detailed embodiments of the present invention are disclosed herein; it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limits, but merely as a basis for teaching one skilled in the art to employ the present invention. The specific examples below will enable the invention to be better understood. However, they are given merely by way of guidance and do not imply any limitation.
The present invention may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this invention is not limited to the specific materials, devices, methods, applications, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention. The term “plurality”, as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.
It is to be appreciated that certain features of the invention which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. Further reference to values stated in ranges includes each and every value within that range.
The following definitions are intended to assist in understanding the present invention:
The term “biocompatible” is defined to mean the sorbent is capable of coming in contact with physiologic fluids, living tissues, or organisms, without producing unacceptable clinical changes during the time that the sorbent is in contact with the physiologic fluids, living tissues, or organisms.
The term “hemocompatible” is defined as a condition whereby a biocompatible material when placed in contact with whole blood or blood plasma results in clinically acceptable physiologic changes.
As used herein, the term “physiologic fluids” are liquids that originate from the body and can include, but are not limited to, nasopharyngeal, oral, esophageal, gastric, pancreatic, hepatic, pleural, pericardial, peritoneal, intestinal, prostatic, seminal, vaginal secretions, as well as tears, saliva, lung, or bronchial secretions, mucus, bile, blood, lymph, plasma, serum, synovial fluid, cerebrospinal fluid, urine, and interstitial, intracellular, and extracellular fluid, such as fluid that exudes from burns or wounds.
As used herein, the term “laboratory or manufacturing fluids” are defined as liquids that are used in life sciences applications that include, but are not limited to, tissue and cell culture media and additives, chemical or biologic assay media, sample preparation buffers, biologic manufacturing media, growth media, and bioreactor media.
As used herein, the term “sorbent” includes adsorbents and absorbents.
For purposes of this invention, the term “sorb” is defined as “taking up and binding by absorption and adsorption”.
For the purposes of this invention, the term “perfusion” is defined as passing a physiologic fluid, once through or by way of a suitable extracorporeal circuit, through a device containing the porous polymeric adsorbent to remove toxic molecules from the fluid.
The term “hemoperfusion” is a special case of perfusion where the physiologic fluid is blood.
The term “dispersant” or “dispersing agent” is defined as a substance that imparts a stabilizing effect upon a finely divided array of immiscible liquid droplets suspended in a fluidizing medium.
The term “heparin mimicking polymer” refers to any polymer that possesses the same anticoagulant and/or antithrombogenic properties as heparin.
The term “macroreticular synthesis” is defined as a polymerization of monomers into polymer in the presence of an inert precipitant which forces the growing polymer molecules out of the monomer liquid at a certain molecular size dictated by the phase equilibria to give solid nanosized microgel particles of spherical or almost spherical symmetry packed together to give a bead with physical pores of an open cell structure [U.S. Pat. No. 4,297,220, Meitzner and Oline, Oct. 27, 1981; R. L. Albright, Reactive Polymers, 4, 155-174(1986)].
The term “hypercrosslinked” describes a polymer in which the single repeating unit has a connectivity of more than two. Hypercrosslinked polymers are prepared by crosslinking swollen, or dissolved, polymer chains with a large number of rigid bridging spacers, rather than copolymerization of monomers. Crosslinking agents may include bis(chloromethyl) derivatives of aromatic hydrocarbons, methylal, monochlorodimethyl ether, and other bifunctional compounds that react with the polymer in the presence of Friedel-Crafts catalysts [Tsyurupa, M. P., Z. K. Blinnikova, N. A. Proskurina, A. V. Pastukhov, L. A. Pavlova, and V. A. Davankov. “Hypercrosslinked Polystyrene: The First Nanoporous Polymeric Material.” Nanotechnologies in Russia 4 (2009): 665-75.]
Some preferred polymers comprise residues from one or more monomers, or containing monomers, or mixtures thereof, selected from acrylonitrile, allyl ether, allyl glycidyl ether, butyl acrylate, butyl methacrylate, cetyl acrylate, cetyl methacrylate, 3,4-dihydroxy-1-butene, dipentaerythritol diacrylate, dipentaerythritol dimethacrylate, dipentaerythritol tetraacrylate, dipentaerythritol tetramethacrylate, dipentaerythritol triacrylate, dipentaerythritol trimethacrylate, divinylbenzene, divinylformamide, divinylnaphthalene, divinylsulfone, 3,4-epoxy-1-butene, 1,2-epoxy-9-decene, 1,2-epoxy-5-hexene, ethyl acrylate, ethyl methacrylate, ethylstyrene, ethylvinylbezene, glycidyl methacrylate, methyl acrylate, methyl methacrylate, octyl acrylate, octyl methacrylate, pentaerythritol diacrylate, pentaerythritol dimethacrylate, pentaerythritol tetraacrylate, pentaerythritol tetramethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, styrene, trimethylolpropane diacrylate, trimethylolpropane dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, trivinylbenzene, trivinylcyclohexane, vinyl acetate, vinylbenzyl alcohol, 4-vinyl-1-cyclohexene 1,2-epoxide, vinylformamide, vinylnaphthalene, 2-vinyloxirane, and vinyltoluene.
Some embodiments of the invention use an organic solvent and/or polymeric porogen as the porogen or pore-former, and the resulting phase separation induced during polymerization yield porous polymers. Some preferred porogens are selected from, or mixtures comprised of any combination of, benzyl alcohol, cyclohexane, cyclohexanol, cyclohexanone, decane, dibutyl phthalate, di-2-ethylhexyl phthalate, di-2-ethylhexylphosphoric acid, ethylacetate, 2-ethyl-1-hexanoic acid, 2-ethyl-1-hexanol, n-heptane, n-hexane, isoamyl acetate, isoamyl alcohol, n-octane, pentanol, poly(propylene glycol), polystyrene, poly(styrene-co-methyl methacrylate), tetraline, toluene, tri-n-butylphosphate, 1,2,3-trichloropropane, 2,2,4-trimethylpentane, xylene.
In yet another embodiment, the dispersing agent is selected from a group consisting of hydroxyethyl cellulose, hydroxypropyl cellulose, poly(diethylaminoethyl acrylate), poly(diethylaminoethyl methacrylate), poly(dimethylaminoethyl acrylate), poly(dimethylaminoethyl methacrylate), poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate), poly(hydroxypropyl acrylate), poly(hydroxypropyl methacrylate), poly(vinyl alcohol), salts of poly(acrylic acid), salts of poly(methacrylic acid) and mixtures thereof.
Preferred sorbents are biocompatible. In another further embodiment, the polymer is biocompatible. In yet another embodiment, the polymer is hemocompatible. In still a further embodiment, the biocompatible polymer is hemocompatible. In still a further embodiment, the geometry of the polymer is a spherical bead.
In another embodiment, the biocompatible polymer comprises poly(N-vinylpyrrolidone).
The coating/dispersant on the porous poly(styrene-co-divinylbenzene) resin will imbue the material with improved biocompatibility.
In still yet another embodiment, a group of cross-linkers consisting of dipentaerythritol diacrylates, dipentaerythritol dimethacrylates, dipentaerythritol tetraacrylates, dipentaerythritol tetramethacrylates, dipentaerythritol triacrylates, dipentaerythritol trimethacrylates, divinylbenzene, divinylformamide, divinylnaphthalene, divinylsulfone, pentaerythritol diacrylates, pentaerythritol dimethacrylates, pentaerythritol tetraacrylates, pentaerythritol tetramethacrylates, pentaerythritol triacrylates, pentaerythritol trimethacrylates, trimethylolpropane diacrylate, trimethylolpropane dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, trivinylbenzene, trivinylcyclohexane and mixtures thereof can be used in formation of a hemocompatible hydrogel coating.
In some embodiments, the polymer is a polymer comprising at least one crosslinking agent and at least one dispersing agent. The dispersing agent may be biocompatible. The dispersing agents can be selected from chemicals, compounds or materials such as hydroxyethyl cellulose, hydroxypropyl cellulose, poly(diethylaminoethyl acrylate), poly(diethylaminoethyl methacrylate), poly(dimethylaminoethyl acrylate), poly(dimethylaminoethyl methacrylate), poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate), poly(hydroxypropyl acrylate), poly(hydroxypropyl methacrylate), poly(vinyl alcohol), salts of poly(acrylic acid), salts of poly(methacrylic acid) and mixtures thereof the crosslinking agent selected from a group consisting of dipentaerythritol diacrylates, dipentaerythritol dimethacrylates, dipentaerythritol tetraacrylates, dipentaerythritol tetramethacrylates, dipentaerythritol triacrylates, dipentaerythritol trimethacrylates, divinylbenzene, divinylformamide, divinylnaphthalene, divinyl sulfone, pentaerythritol diacrylates, pentaerythritol dimethacrylates, pentaerythritol tetraacrylates, pentaerythritol tetramethacrylates, pentaerythritol triacrylates, pentaerythritol trimethacrylates, trimethylolpropane diacrylate, trimethylolpropane dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, trivinylbenzene, trivinylcyclohexane and mixtures thereof. Preferably, the polymer is developed simultaneously with the formation of the coating, wherein the dispersing agent is chemically bound or entangled on the surface of the polymer.
In still another embodiment, the biocompatible polymer coating is selected from a group consisting of poly(hydroxyethyl methacrylate), poly(hydroxyethyl acrylate), poly(dimethylaminoethyl methacrylate), salts of poly(acrylic acid), salts of poly(methacrylic acid), poly(diethylaminoethyl methacrylate), poly(hydroxypropyl methacrylate), poly(hydroxypropyl acrylate), poly(N-vinylpyrrolidone), poly(vinyl alcohol) and mixtures thereof. In another embodiment, the salts may be sodium and potassium salts and in still another embodiment, the salts are water-soluble salts.
In still another embodiment, the biocompatible oligomer coating is selected from a group consisting of poly(hydroxyethyl methacrylate), poly(hydroxyethyl acrylate), poly(dimethylaminoethyl methacrylate), salts of poly(acrylic acid), salts of poly(methacrylic acid), poly(diethylaminoethyl methacrylate), poly(hydroxypropyl methacrylate), poly(hydroxypropyl acrylate), poly(N-vinylpyrrolidone), poly(vinyl alcohol) and mixtures thereof. In another embodiment, the salts may be sodium and potassium salts and in still another embodiment, the salts are water-soluble salts.
The present biocompatible sorbent compositions are comprised of a plurality of pores. The biocompatible sorbents are designed to adsorb a broad range of toxins from less than 0.5 kDa to 1,000 kDa. While not intending to be bound by theory, it is believed the sorbent acts by sequestering molecules of a predetermined molecular weight within the pores. The size of a molecule that can be sorbed by the polymer will increase as the pore size of the polymer increases. Conversely, as the pore size is increased beyond the optimum pore size for adsorption of a given molecule, adsorption of said protein may or will decrease.
In certain methods, the solid form is porous. Some solid forms are characterized as having a pore structure having a total volume of pore sizes in the range of from 10 Å to 40,000 Å greater than 0.1 cc/g and less than 5.0 cc/g dry polymer.
In certain embodiments, the polymers can be made in bead form having a diameter in the range of 0.1 micrometers to 2 centimeters. Certain polymers are in the form of powder, beads or other regular or irregularly shaped particulates.
In some embodiments, the plurality of solid forms comprises particles having a diameter in the range for 0.1 micrometers to 2 centimeters.
In some methods, the undesirable molecules are inflammatory mediators and stimulators comprised of cytokines, superantigens, monokines, chemokines, interferons, proteases, enzymes, peptides including bradykinin, soluble CD40 ligand, bioactive lipids, oxidized lipids, cell-free hemoglobin, damage-associated molecular pattern (DAMPs), Pathogen-associated molecular pattern molecules (PAMPs), cell-free myoglobin, growth factors, glycoproteins, prions, toxins, bacterial and viral toxins, endotoxins, drugs, vasoactive substances, foreign antigens, antibodies, and positively charged ions, including, but not limited to, potassium.
In some methods, the antibodies can be immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), immunoglobulin D (IgG), immunoglobulin D (IgM) and/or immunoglobulin fragments or subunits.
DAMPs have been associated with countless syndromes and diseases. These include complications from trauma, burns, traumatic brain injury and invasive surgery, and also organ-specific illnesses like liver disease, kidney dialysis complications, and autoimmune diseases. DAMPs are host molecules that can initiate and perpetuate noninfectious SIRS and exacerbate infectious SIRS. DAMPs are a diverse family of molecules that are intracellular in physiological conditions and many are nuclear or cytosolic proteins. DAMPs can be divided into two groups: (1) molecules that perform noninflammatory functions in living cells (such as HMGB1) and acquire immunomodulatory properties when released, secreted, modified, or exposed on the cell surface during cellular stress, damage, or injury, or (2) alarmins, i.e., molecules that possess cytokine-like functions (such as β-Defensins and Cathelicidin), which can be stored in cells and released upon cell lysis, whereupon they contribute to the inflammatory response. When released outside the cell or exposed on the surface of the cell following tissue injury, they move from a reducing to an oxidizing milieu, which affects their activity. Also, following necrosis, mitochondrial and nuclear DNA fragments are released outside the cell becoming DAMPs.
DAMPs, such as HMGB-1, heat-shock and S100 proteins are normally found inside cells and are released by tissue damage. DAMPs act as endogenous danger signals to promote and exacerbate the inflammatory response. HMGB-1 is a non-histone nuclear protein that is released under stress conditions. Extracellular HMGB-1 is an indicator of tissue necrosis and has been associated with an increased risk of sepsis and multiple organ dysfunction syndrome (MODS). S100 A8 (granulin A, MRP8) and A9 (granulin B\, MRP14) homo and heterodimers bind to and signal directly via the TLR4/lipopolysaccharide receptor complex where they become danger signals that activate immune cells and vascular endothelium. Procalcitonin is a marker of severe sepsis caused by bacteria and its release into circulation is indicative of the degree of sepsis. Serum amyloid A (SAA), an acute-phase protein, is produced predominantly by hepatocytes in response to injury, infection, and inflammation. During acute inflammation, serum SAA levels may rise by 1000-fold. SAA is chemotactic for neutrophils and induces the production of proinflammatory cytokines. Heat shock proteins (HSP) are a family of proteins that are produced by cells in response to exposure to stressful conditions and are named according to their molecular weight (10, 20-30, 40, 60, 70, 90). The small 8-kilodalton protein ubiquitin, which marks proteins for degradation, also has features of a heat shock protein. Hepatoma-derived growth factor (HDGF), despite its name, is a protein expressed by neurons. HDGF can be released actively by neurons via a nonclassical pathway and passively by necrotic cells. Other factors, such as complement factors 3 and 5, are activated as part of the host defense against pathogens but can also contribute to the adverse outcomes in sepsis. Excessive, persistent circulating levels of cytokines and DAMPs contribute to organ injury and identify those patients who have the highest risk of multiple organ dysfunction (MODs) and death in community acquired pneumonia and sepsis.
PAMPs include lipopolysaccharides, lipopeptides, lipoteichoic acid, peptidoglycans, nucleic acids such as double-stranded RNA, toxins and flagellins nd can trigger an immune response in the host (e.g. the innate immune system) to fight the infection, leading to the production of high levels of inflammatory and anti-inflammatory mediators, such as cytokines. PAMPs and high cytokine levels, as well as direct tissue injury (trauma, burns, etc.), can damage tissue, causing the extracellular release of damage-associated molecular pattern (DAMPs) molecules into the bloodstream. DAMPs are a broad class of endogenous molecules, which like PAMPs, trigger the immune response through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs).
Preferred sorbents include cross-linked polymeric material derived from the reaction of a cross-linker with one or more of the following polymerizable monomers, then subsequently sulfonated to form a sulfonic acid salt: acrylonitrile, butyl acrylate, butyl methacrylate, cetyl acrylate, cetyl methacrylate, divinylbenzene, ethyl acrylate, ethyl methacrylate, ethylstyrene, methyl acrylate, methyl methacrylate, octyl acrylate, octyl methacrylate, styrene, vinylbenzyl alcohol, vinylformamide, vinylnaphthalene, or vinyltoluene.
In some embodiments, radically polymerizable vinyl monomers containing ˜SO3Na groups, or ˜SO3H groups, can be used in graft copolymerization with porous polymers containing polymerizable double bonds. These monomers can be selected from 4-styrene sulfonic acid sodium salt, vinyl sulfonic acid sodium salt, 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-acrylamido-2-methyl-1-propanesulfonic sodium salt, 3-sulfopropyl acrylate sodium salt, 3-sulfopropyl methacrylate sodium salt, [2-(methacryloyloxy)ethyl] dimethyl-(3-sulfopropyl)ammonium hydroxide, N-(3-sulfopropyl)-N-(methacryl oxy ethyl)-N,N-dimethyl ammonium betaine, para-styrene sulfonyl chloride, or any combinations thereof. Furthermore, para-styrene sulfonyl chloride can be polymerized in the presence of divinylbenzene and hydrolyzed with sodium hydroxide solution to directly yield poly(styrene-co-divinylbenzene) porous material with ˜SO3Na groups.
In another embodiment, the present invention relates to a sulfonated polymer comprised of at least one crosslinking agent for making the polymer and at least one dispersing agent whereby the dispersing agent forms a biocompatible surface on the polymer.
In one embodiment the porous polymers of this invention are made by suspension polymerization in a formulated aqueous phase with free radical initiation in the presence of aqueous phase dispersants that are selected to provide a biocompatible and a hemocompatible exterior surface to the formed polymer beads. The sulfonation of the resultant beads yields an ion exchange resin coated with a hemocompatible hydrogel. The beads are made porous by the macroreticular synthesis with an appropriately selected porogen (pore forming agent) and an appropriate time-temperature profile for the polymerization in order to develop the proper pore structure. The subsequent introduction of the sulfonic acid groups in the already formed network forms a sulfonic acid salt inner core (ion exchange resin) and a hemocompatible outer hydrogel exterior. Suitable choice of the reaction conditions for the sulfonation allows preservation or expression (via a protecting group) of the hemocompatible nature of the exterior hydrogel.
In another embodiment polymers made by suspension polymerization can be made biocompatible and hemocompatible by further grafting of biocompatible and hemocompatible monomers or low molecular weight oligomers. It has been shown that the radical polymerization procedure does not consume all the vinyl groups of DVB introduced into copolymerization. On average, about 30% of DVB species fail to serve as crosslinking bridges and remain involved in the network by only one of two vinyl groups. The presence of a relatively high amount of pendant vinyl groups is therefore a characteristic feature of the macroporous adsorbents. It can be expected that these pendant vinyl groups are preferably exposed to the surface of the polymer beads and their macropores should be readily available to chemical modification. The chemical modification of the surface of macroporous DVB-copolymers relies on chemical reactions of the surface-exposed pendant vinyl groups and aims at converting these groups into more hydrophilic functional groups. This conversion via free radical grafting of monomers and/or cross-linkers or low molecular weight oligomers provides the initial hydrophobic adsorbing material with the property of hemocompatibility. The subsequent introduction of the sulfonic acid groups into the already formed network forms a sulfonic acid salt inner core (ion exchange resin) and a hemocompatible outer hydrogel exterior. Suitable choice of the reaction conditions for the sulfonation allows preservation or expression (via a protecting group) of the hemocompatible nature of the exterior hydrogel.
Still another embodiment consists of binding long hydrophilic polymer chains onto the beads' surfaces, which should preclude contact between blood cells and the sulfonated polystyrene surface. This can be accomplished via free radical or SN2 type chemistry. The chemical modification of the surface of sorbent beads, which is the case in the above modification, is facilitated by the remarkable peculiarity of the hypercrosslinked polystyrene; namely, that the reactive functional groups of the polymer are predominantly located on its surface. The hypercrosslinked polystyrene is generally prepared by crosslinking polystyrene chains with large amounts of bifunctional compounds, in particular, those bearing two reactive chloromethyl groups. The latter alkylate, in a two-step reaction, two phenyl groups of neighboring polystyrene chains according to Friedel-Crafts reaction, with evolution of two molecules of HCl and formation of a cross bridge. During the crosslinking reaction, the three-dimensional network formed acquires rigidity. This property gradually reduces the rate of the second step of the crosslinking reaction, since the reduced mobility of the second pendant functional group of the initial crosslinking reagent makes it more and more difficult to add an appropriate second partner for the alkylation reaction. This is especially characteristic of the second functional groups that happen to be exposed to the surface of the bead. Therefore, of the pendant unreacted chloromethyl groups in the final hypercrosslinked polymer, the largest portion, if not the majority of the groups, are located on the surface of the bead (or on the surface of large pores). This circumstance makes it possible to predominantly modify the surface of the polymer beads by involving the above chloromethyl groups into various chemical reactions that allow attachment of biocompatible and hemocompatible monomers, and/or cross-linkers or low molecular weight oligomers. The subsequent introduction of the sulfonic acid groups in the already formed network forms a sulfonic acid salt inner core (ion exchange resin) and a hemocompatible outer hydrogel exterior. Suitable choice of the reaction conditions for the sulfonation allows preservation or expression (via a protecting group) of the hemocompatible nature of the exterior hydrogel.
In yet another embodiment, the radical polymerization initiator is initially added to the dispersed organic phase, not the aqueous dispersion medium as is typical in suspension polymerization. During polymerization, many growing polymer chains with their chain-end radicals show up at the phase interface and can initiate the polymerization in the dispersion medium. Moreover, the radical initiator, like benzoyl peroxide, generates radicals relatively slowly. This initiator is only partially consumed during the formation of beads even after several hours of polymerization. This initiator easily moves toward the surface of the bead and activates the surface exposed pendant vinyl groups of the divinylbenzene moiety of the bead, thus initiating the graft: polymerization of other monomers added after the reaction has proceeded for a period of time. Therefore, free-radical grafting can occur during the transformation of the monomer droplets into polymer beads thereby incorporating monomers and/or cross-linkers or low molecular weight oligomers that impart biocompatibility or hemocompatibility as a surface coating. The subsequent introduction of the sulfonic acid groups in the already formed network forms a sulfonic acid salt inner core (ion exchange resin) and a hemocompatible outer hydrogel exterior. Suitable choice of the reaction conditions for the sulfonation allows preservation or expression (via a protecting group) of the hemocompatible nature of the exterior hydrogel.
In still yet another embodiment, hypercrosslinked or macroreticular porous polymer beads (including commercial versions) that have been sulfonated under mild conditions that retain residual functionality such as unreacted double bonds or chloromethyl groups can be modified via free radical or SN2 type chemistry which would allow attachment of biocompatible and a hemocompatible monomers, and/or cross-linkers or low molecular weight oligomers. Among various “mild” sulfonating agents, Acetyl Sulfate (prepared from 98% conc. Sulfuric acid and acetic anhydride at low temperatures) is known to be very efficient for DVB or Styrene based polymeric materials. Sulfonation is usually done at 50° C. for several hours using equimolar amounts of acetyl sulfate and DVB or styrene based polymers. Sulfonation occurs mainly at the benzene ring and unreacted double bonds (in DVB based cross-linked polymeric porous beads) would be preserved for further functionalization. Usually after sulfonation with acetyl sulfate, the polymer is converted into —SO3Na form and can be graft copolymerized with N-vinyl pyrrolidone or other hemocompatible monomers and/or cross-linkers or low molecular weight oligomers (in bulk with benzoyl peroxide as initiator) or in water solutions (using sodium persulfate initiator). Resulting sulfonated polymer is “coated” with poly(N-vinylpyrrolidone), as an example, to create a hemocompatible material capable of removing K+ cations from physiological fluids.
Some embodiments of the invention involve direct synthesis of porous polymeric beads containing —SO3Na groups. Any polymerizable vinyl monomer containing —SO3Na (or —SO3H) groups can be polymerized in the presence of cross-linker monomer (like DVB, bis-acrylamide, bis-(meth)acrylates, etc.) and suitable porogen to yield porous polymeric beads containing above mentioned functionalities (—SO3Na or SO3H). Vinyl monomers containing SO3Na or SO3H groups can also be copolymerized with hemocompatible monomer (NVP. 2-HEMA, etc.) in presence of porogen to yield hemocompatible porous beads containing —SO3Na groups.
Another embodiment of the invention involves making porous polymer beads containing SO3Na groups via graft copolymerization of premade porous polymers (containing double bonds unreacted) with polymerizable vinyl monomers containing —SO3Na or —SO3H groups (with the mixture of suitable hemocompatible vinyl monomers). Such monomers can be vinyl sulfonic acid Na salt, 4-styrene sulfonic acid Na salt, etc.
The hemoperfusion and perfusion devices consist of a packed bead bed of the porous polymer beads in a flow-through container fitted with either a retainer screen at both the exit end and the entrance end to maintain the bead bed inside the container or with a subsequent retainer screen to collect the beads after mixing. The hemoperfusion and perfusion operations are performed by passing the whole blood, blood plasma or physiologic fluid through the packed bead bed. During the perfusion through the bead bed, the toxic molecules are retained by sorption, torturous path, and/or ion exchange mechanism the while the remainder of the fluid and intact cell components pass through essentially unchanged in concentration.
In some other embodiments, an in-line filter is comprised of a packed bead bed of the porous polymer beads in a flow-through container, fitted with a retainer screen at both the exit end and the entrance end to maintain the bead bed inside the container. pRBCs are passed from a storage bag once-through the packed bead bed via gravity, during which the toxic molecules are retained by sorption, torturous path, and/or ion exchange mechanisms, while the remainder of the fluid and intact cell components pass through essentially unchanged in concentration.
Certain polymers useful in the invention (as is or after further modification) are macroporous polymers prepared from the polymerizable monomers of styrene, divinylbenzene, ethylvinylbenzene, and the acrylate and methacrylate monomers such as those listed below by manufacturer. Rohm and Haas Company, (now part of Dow Chemical Company): macroporous polymeric sorbents such as Amberlite™ XAD-1, Amberlite™ XAD-2, Amberlite™ XAD-4, Amberlite™ XAD-7, Amberlite™ XAD-7HP, Amberlite™ XAD-8, Amberlite™ XAD-16, Amberlite™ XAD-16 HP, Amberlite™ XAD-18, Amberlite™ XAD-200, Amberlite™ XAD-1180, Amberlite™ XAD-2000, Amberlite™ XAD-2005, Amberlite™ XAD-2010, Amberlite™ XAD-761, and Amberlite™ XE-305, and chromatographic grade sorbents such as Amberchrom™ CG 71,s,m,c, Amberchrom™ CG 161,s,m,c, Amberchrom™ CG 300,s,m,c, and Amberchrom™ CG 1000,s,m,c. Dow Chemical Company: Dowex™ Optipore™ L-493, Dowex™ Optipore™ V-493, Dowex™ Optipore™ V-502, Dowex™ Optipore™ L-285, Dowex™ Optipore™ L-323, and Dowex™ Optipore™ V-503. Lanxess (formerly Bayer and Sybron): Lewatit™ VPOC 1064 MD PH, Lewatit™ VPOC 1163, Lewatit™ OC EP 63, Lewatit™ S 6328A, Lewatit™ OC 1066, and Lewatit™ 60/150 MIBK. Mitsubishi Chemical Corporation: Diaion™ HP 10, Diaion™ HP 20, Diaion™ HP 21, Diaion™ HP 30, Diaion™ HP 40, Diaion™ HP 50, Diaion™ SP70, Diaion™ SP 205, Diaion™ SP 206, Diaion™ SP 207, Diaion™ SP 700, Diaion™ SP 800, Diaion™ SP 825, Diaion™ SP 850, Diaion™ SP 875, Diaion™ HP 1MG, Diaion™ HP 2MG, Diaion™ CHP 55A, Diaion™ CHP 55Y, Diaion™ CHP 20A, Diaion™ CHP 20Y, Diaion™ CHP 2MGY, Diaion™ CHP 20P, Diaion™ HP 20SS, Diaion™ SP 20SS, Diaion™ SP 207SS. Purolite Company: Purosorb™ AP 250 and Purosorb™ AP 400, and Kaneka Corp. Lixelle beads.
Various proteins may be adsorbed by the composition of the instant disclosure. Some of these proteins and their molecular weights are shown in the table below.
Molecular
Protein
Weight (Da)
PAF (Platelet Activating Factor)
524
bilirubin
548.6
heme b
616.5
MIP-1alpha
8,000
Complement C5a
8,200
Complement C3a
9,089
IL-8
9,000
S100B (dimerizes)
10,000
β-2 microglobulin
11,800
Procalcitonin
13,000
Phospholipase A2, secretory PLA2 type I
14,000
pancreatic
PLA2G2A
16,083
IL-7
17,400
Myoglobin
17,699
Trypsin-human pancreas
23,300
IL-6
23,718
Toxic shock syndrome toxin 1 (TSST-1
24,000
Enterotoxin B, S aureus
24,500
HMGB1
24,894
Interferon gamma
25,000
Chymotrypsin
25,000
Elastase (neutrophil)
25,000
Trypsin
26,488
PF4
27,100
Enterotoxin A, S. aureus
27,800
alpha toxin A&B, S. aureus
28,000
PCNA, proliferating cell nuclear antigen
29,000
Arginse I
35,000
Carboxypeptidase A
35,000
Thrombin
36,700
alpha-1 antitrypsin
44,324
TNF-alpha
52,000
Activated Protein C
56,200
Amylase
57,000
hemopexin
57,000
alpha-1 antichymotrypsin
55,000-68,000
Diptheria toxoid
62,000
hemoglobin, oxy
64,000
Pseudomonas Exotoxin A
66,000
ShigaToxin (A 32 kDa, 5 × B 7.7 kDa)
69,000
Calpain-1 (human erythrocytes)
112,00
C reactive Protein (5 × 25 kDa)
115,000
Myeloperoxidase (neutrophils)
150,000
Immunoglobulin G IgG
150,000
NOS synthase
150,000
Immunoglobulin A IgA
162,000
Immunoglobulin E (IgE)
190,000
Immunoglobulin M IgM
950,000
The following examples are intended to be exemplary and non-limiting.
The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.
Reactor Setup; a 4-neck glass lid was affixed to a 3000 mL jacketed cylindrical glass reaction vessel using a stainless steel flange clamp and PFTE gasket. The lid was fitted with a PFTE stirrer bearing, RTD probe adapter, and water-cooled reflux condenser. A stainless steel stirring shaft having five 45° agitators was fit through the stirrer bearing and inserted into a digital overhead stirrer. An RTD probe was fit through the corresponding adapter, and connected to a PolyStat circulating heating and chilling unit. Compatible tubing was used to connect the inlet and outlet of the reaction vessel jacket to the appropriate ports on the PolyStat. The unused port in the lid was used for charging the reactor and was plugged at all other times.
Polymerization; Aqueous phase and organic phase compositions are shown below, in Table I and Table II, respectively. Ultrapure water was split into approximately equal parts in two separate Erlenmeyer flasks each containing a PFTE coated magnetic stir bar. Poly(vinyl alcohol) (PVA), having a degree of hydrolysis of 85.0 to 89.0 mol percent and a viscosity of 23.0 to 27.0 cP in a 4% aqueous solution at 20° C., was dispersed into the water in the first flask and heated to 80° C. on a hot plate with agitation. Salts (see Table 1, MSP, DSP, TSP and Sodium nitrite) were dispersed into the water in the second flask and heated to 80° C. on a hot plate with agitation. Circulation of heat transfer fluid from the PolyStat through the reaction vessel jacket was started, and fluid temperature heated to 60° C. Once PVA and salts dissolved, both solutions were charged to the reactor, one at a time, using a glass funnel. The digital overhead stirrer was powered on and the rpm set to a value to form appropriate droplet sizes upon organic phase addition. Temperature of the aqueous phase in the kettle was set to 70° C. The organic phase was prepared by adding benzoyl peroxide (BPO) to the divinylbenzene (DVB) and styrene in a 2 L Erlenmeyer flask and swirling until completely dissolved. 2,2,4-trimethylpentane and toluene were added to the flask, which was swirled to mix well. Once the temperature of the aqueous phase in the reactor reached 70° C., the organic phase was charged into the reactor using a narrow-necked glass funnel. Temperature of the reaction volume dropped upon the organic addition. A temperature program for the PolyStat was started, heating the reaction volume from 60 to 77° C. over 30 minutes, 77 to 80° C. over 30 minutes, holding the temperature at 80° C. for 960 minutes, and cooling to 20° C. over 60 minutes. 1-Vinyl-2-pyrrolidinone (VP) was added dropwise via glass separatory funnel once the reaction reached identity point, approximately one hour after the reaction temperature reached 80° C. Note: the temperature program for preparation of polymer CY15042 was different, proceeding as follows; reaction volume heated from 55 to 62° C. over 30 minutes, 62 to 65° C. over 30 minutes, held at 65° C. for 1320 minutes, heated from 65 to 82° C. over 30 minutes, 82 to 85° C. over 30 minutes, held at 85° C. for 60 minutes, then cooled to 20° C. over 60 minutes.
TABLE I
Aqueous Phase Composition
Reagent
Mass (g)
Ultrapure water
1500.000
Poly(vinyl alcohol) (PVA)
4.448
Monosodium phosphate (MSP)
4.602
Disodium phosphate (DSP)
15.339
Trisodium phosphate (TSP)
9.510
Sodium nitrite
0.046
Total
1533.899
TABLE II
Organic Phase Compositions
CY12004
CY15042
CY15044
CY15045
CY15077
Reagent
Mass (g)
Mass (g)
Mass (g)
Mass (g)
Mass (g)
Divinylbenzene, 63% (DVB)
508.751
451.591
386.284
386.284
498.383
Styrene
0.000
0.00
374.118
374.118
0.000
2,2,4-trimethylpentane (Isooctane)
384.815
125.800
271.210
271.210
482.745
Toluene
335.004
712.869
235.725
235.725
222.404
Benzoyl peroxide, 98% (BPO)
3.816
18.432
5.703
5.703
3.738
1-Vinyl-2-pyrrolidinone (VP)
151.578
0.000
0.000
167.288
0.000
Total (excluding BPO and VP)
1228.571
1290.260
1267.337
1267.337
1203.532
The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.
Work-up; reaction volume level in the reactor was marked. Overhead stirrer agitation was stopped, residual liquid siphoned out of the reactor, and the reactor filled to the mark with ultrapure water at room temperature. Overhead stirrer agitation was restarted and the slurry heated to 70° C. as quickly as possible. After 30 minutes, agitation was stopped and residual liquid siphoned out. Polymer beads were washed five times in this manner. During the final wash, the slurry temperature was cooled to room temperature. After the final water wash, polymer beads were washed with 99% isopropyl alcohol (IPA) in the same manner. 99% IPA was siphoned out and replaced with 70% IPA before transferring the slurry into a clean 4 L glass container. Unless noted otherwise, on an as-needed basis the polymer was steam stripped in a stainless steel tube for 8 hours, rewet in 70% IPA, transferred into DI water, sieved to obtain only the portion of beads having diameters between 300 and 600 μm, and dried at 100° C. until no further weight loss on drying was observed.
Cumulative pore volume data, measured by nitrogen desorption isotherm, for polymers CY12004, CY15042, CY15044, and CY15045, are presented below, in Tables III, IV, V, and VI, respectively. Cumulative pore volume data, measured by mercury intrusion porosimetry, for polymer CY15077 is presented in Table VII, below.
TABLE III
Nitrogen Desorption Data for CY12004
Pore Diameter
Average
Cumulative Pore
Range (Å)
Diameter (Å)
Volume (cm3/g)
1221.6-868.1
985.2149834
0.009113091
868.1-751.9
801.4105771
0.019081821
751.9-661.5
700.749642
0.032021618
661.5-613.5
635.6650389
0.048206769
613.5-568.5
589.2088599
0.067981224
568.5-509.8
535.8385194
0.114704165
509.8-456.1
479.8625277
0.214714265
456.1-418.7
435.7117054
0.311269356
418.7-374.6
394.0534583
0.455991378
374.6-330.2
349.456374
0.579735461
330.2-319.6
324.7147611
0.612988132
319.6-281.8
298.1620033
0.708072633
281.8-273.9
277.7142728
0.73291244
273.9-256.6
264.6494358
0.777049805
256.6-237.0
245.9517985
0.830089884
237.0-225.7
231.0229263
0.857298007
225.7-215.6
220.375968
0.88145223
215.6-145.5
166.3375231
1.066971104
145.5-104.6
117.8539174
1.181204175
104.6-84.4
92.0541661
1.241569291
84.4-71.4
76.67121175
1.285618005
71.4-60.9
65.20679768
1.326059561
60.9-52.7
56.07123392
1.360787093
52.7-46.5
49.12518253
1.389258246
46.5-41.3
43.53851295
1.416541075
41.3-37.1
38.91936166
1.445235862
TABLE IV
Nitrogen Desorption Data for CY15042
Pore Diameter
Average
Cumulative Pore
Range (Å)
Diameter (Å)
Volume (cm3/g)
2011.0-633.1
751.380276
0.003621266
633.1-424.8
488.0919378
0.006317461
424.8-418.5
421.593936
0.006912678
418.5-353.5
380.29179
0.008267096
353.5-280.4
308.2300243
0.011094129
280.4-275.4
277.8814342
0.01168737
275.4-249.5
261.1230419
0.012721633
249.5-209.1
225.543664
0.015611261
209.1-206.8
207.9070897
0.016388077
206.8-137.5
157.790999
0.442556595
137.5-98.1
110.7933773
0.765560391
98.1-81.8
88.28728758
0.845836735
81.8-67.3
72.96250925
0.911182647
67.3-57.7
61.63744463
0.954008444
57.7-50.3
53.43111186
0.983515641
50.3-44.4
46.93705679
1.010486042
44.4-38.6
41.02620024
1.037817277
38.6-34.5
36.30857144
1.058861412
34.5-30.9
32.48566551
1.08400665
30.9-27.3
28.85395017
1.10131894
27.3-24.3
25.59611525
1.12576046
24.3-22.3
23.18199338
1.143118464
22.3-19.6
20.72009386
1.167009752
19.6-17.4
18.32182238
1.190109864
TABLE V
Nitrogen Desorption Data for CY15044
Pore Diameter
Average
Cumulative Pore
Range (Å)
Diameter (Å)
Volume (cm3/g)
2529.6-789.0
936.1742201
1.75877E−06
789.0-446.0
526.203721
0.000135623
446.0-219.6
260.7379647
0.002068559
219.6-213.4
216.3756282
0.004663144
213.4-205.7
209.3598959
0.0088853
205.7-144.7
164.0510277
0.131650053
144.7-99.6
113.2793455
0.294709491
99.6-82.1
88.98089675
0.331539838
82.1-71.4
75.89033961
0.34527909
71.4-60.0
64.52630192
0.360216738
60.0-52.8
55.83732662
0.367929549
52.8-46.8
49.32751384
0.373710394
46.8-41.4
43.66300585
0.378313283
41.4-37.2
39.02724789
0.38481289
37.2-33.2
34.8920748
0.391803441
33.2-30.0
31.34913535
0.393761301
30.0-27.3
28.49102813
0.394422444
27.3-24.7
25.83440471
0.396180539
24.7-22.3
23.34690716
0.401510134
22.3-19.8
20.83368622
0.40782788
19.8-17.5
18.45917969
0.416568116
TABLE VI
Nitrogen Desorption Data for CY15045
Pore Diameter
Average
Cumulative Pore
Range (Å)
Diameter (Å)
Volume (cm3/g)
1277.7-542.6
649.560333
0.000489722
542.6-213.2
252.9981774
0.000667721
213.2-206.9
209.9696024
0.001419558
206.9-141.9
161.6476715
0.261729457
141.9-106.3
118.498425
0.346563251
106.3-84.0
92.17838423
0.37856771
84.0-71.8
76.76600632
0.393497452
71.8-62.4
66.31374327
0.404409264
62.4-53.6
57.17863111
0.411077722
53.6-48.0
50.38372676
0.416000386
48.0-42.5
44.81172299
0.421626585
42.5-38.3
40.08447096
0.428067208
38.3-34.4
36.07215077
0.431303175
34.4-31.5
32.76081107
0.433543649
31.5-26.3
27.29321095
0.440720595
26.3-23.8
24.89263623
0.44207166
23.8-21.3
22.31849785
0.443967237
21.3-19.1
19.99937462
0.45436982
19.1-16.1
17.16801839
0.47745598
TABLE VII
Mercury Intrusion Data for CY15077
Pore size
Cumulative
Diameter (A)
Intrusion (mL/g)
226299.0625
3.40136E−30
213166.0781
0.001678752
201295.1563
0.002518128
172635.8125
0.004364755
139538.0625
0.007554384
113120.7813
0.011919139
90542.36719
0.01645177
78733.25781
0.0203129
72446.375
0.022327403
60340.40234
0.027867284
48343.83984
0.035327822
39009.13672
0.040918175
32136.4082
0.04899035
25330.65625
0.063195683
20981.51563
0.079529688
16219.86426
0.108860672
13252.41211
0.141730919
10501.53613
0.193969816
8359.911133
0.262399256
6786.30127
0.345866203
5538.122559
0.438174427
4337.931152
0.563276172
3501.674805
0.681870878
2838.742188
0.804727197
2593.016846
0.865813017
2266.688965
0.938610673
1831.041748
1.056586146
1509.850708
1.163395643
1394.006104
1.21002543
1294.780151
1.257248282
1207.692627
1.293158531
1131.860962
1.326992273
1065.099976
1.35812819
953.1816406
1.405935764
884.0358887
1.445426106
823.5491333
1.478719592
770.9108276
1.510579824
722.4724731
1.537048101
684.6119995
1.564400196
672.187561
1.581117511
636.7885742
1.60271585
604.7248535
1.621845484
558.1287231
1.651492
518.2624512
1.678913713
483.5536499
1.708594561
453.5110779
1.735918999
426.9998474
1.755934
403.1251526
1.783603072
382.7776794
1.793849826
362.7162476
1.817784309
342.3734436
1.838774562
330.1105042
1.851493955
315.5238037
1.869742155
302.2973938
1.885128617
290.2946777
1.895119786
279.1246643
1.912378907
268.7442627
1.924305081
259.1106873
1.936048627
241.8737793
1.955100656
226.7678223
1.972970247
213.3626251
1.988123298
201.4908142
2.007521152
194.9888611
2.022114754
188.9506989
2.033871174
180.582901
2.035052776
172.8530121
2.050720692
164.9621735
2.062945843
157.8110657
2.071056128
151.1540375
2.082133055
143.9185333
2.096480608
138.4670563
2.106938839
132.8492737
2.119287968
129.5760345
2.126605988
126.5438614
2.126605988
124.2635574
2.132267475
120.8976135
2.141504765
117.3792267
2.150759459
114.791893
2.154810667
111.9475937
2.162935257
108.8830032
2.167646885
106.6480179
2.174062729
104.5217743
2.179908991
102.4295197
2.179908991
100.1580353
2.182951927
98.29322052
2.184018135
96.44822693
2.191127539
94.42159271
2.198545218
91.52587891
2.209161043
89.25807953
2.209312439
87.0777359
2.215425491
85.42358398
2.221472025
83.62612915
2.232139587
82.11174011
2.237514496
79.91614532
2.239231586
78.01462555
2.239560127
76.19993591
2.239560127
75.09249115
2.239560127
73.41201019
2.239560127
72.23709869
2.240245819
71.09960175
2.242422104
69.86301422
2.243849993
68.40761566
2.257676363
67.13697815
2.259181261
66.03359222
2.266284466
65.08189392
2.270181179
64.04368591
2.272682428
62.38490295
2.280714512
61.32764053
2.280714512
60.30379868
2.287917852
59.41370392
2.287917852
58.54679489
2.293802738
57.79866409
2.297607183
56.88977814
2.299046278
55.9213295
2.302111387
54.98665237
2.303381443
The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.
N-vinylpyrrolidone functionalization; base polymer, CY15077, was not steam stripped or sieved prior to functionalization. Two 99% IPA washes at 50° C. were completed during workup for the base polymer, as opposed to one wash at RT. Following IPA washes, the polymer was washed three times with an excess of DI water. Wetted CY15077 polymer beads were added to a 1 L jacketed glass reaction kettle, fitted with a Teflon coated agitator, containing 450 mL DI water, 50.0 g N-vinylpyrrolidone monomer, and 1.5 g sodium persulfate. The reaction was allowed to proceed for 24 hours at 75° C., with agitation speed set to 100 RPM. Upon completion the polymer beads were washed five times with 500 mL DI water at 70° C., steam stripped in a stainless steel tube for 8 hours, rewet in 70% IPA, transferred into DI water, sieved to obtain only the portion of beads having diameters between 300 and 600 μm, and dried at 100° C. until no further weight loss on drying was observed. The yield was 95.5 g of polymer CY15087. Atomic concentrations measured by XPS, and cumulative pore volume data measured by mercury intrusion porosimetry, are shown in Tables VIII and IX, respectively.
TABLE VIII
Atomic Concentrations (in %)
for CY15077 and CY15087
Polymer
Condition
C
N
O
CY15077
Bead
96.2
0.0
3.8
CY15077
Ground
98.6
0.0
1.4
CY15087
Bead
95.5
0.4
4.2
CY15087
Ground
98.3
0.2
1.5
TABLE IX
Mercury Intrusion Data for CY15087
Pore size
Cumulative
Diameter (A)
Intrusion (mL/g)
226275.6875
3.003E−30
213126.625
0.001333927
201250.5938
0.002964283
172601.8438
0.005928566
139532.5469
0.009189277
113124.3359
0.012449989
90545.25
0.015710698
78739.35156
0.017489269
72432.5625
0.01897141
60333.77734
0.021935694
46762.60547
0.026795639
39173.96094
0.03074207
31808.34375
0.034442116
25357.64648
0.040027067
20929.94141
0.046409778
16182.15234
0.056623131
13255.21973
0.065796889
10561.28809
0.080750667
8353.926758
0.105692402
6778.929199
0.138670683
5543.002441
0.177410021
4342.263672
0.24024339
3502.678711
0.308058321
2839.226807
0.388105094
2591.51416
0.428066701
2267.699951
0.48154822
1831.208252
0.570007741
1510.12561
0.655585647
1394.226563
0.696180701
1294.746582
0.729135811
1208.07251
0.76245892
1132.023804
0.795990944
1065.684937
0.815372229
953.989502
0.855566621
883.8703613
0.871785223
823.4996338
0.921781898
771.3513794
0.949763238
722.1901245
1.018806458
684.8914185
1.027466536
671.8579712
1.033001781
636.456604
1.044957519
604.6593018
1.05753231
557.9059448
1.079107881
518.4785156
1.102458835
483.8456726
1.127018452
453.9489746
1.151340365
426.8711243
1.174746156
402.8918152
1.194709539
382.4490967
1.213674426
360.680481
1.231868267
342.5672302
1.252067924
329.8339539
1.267953753
315.4637756
1.28668797
302.4020996
1.299176812
290.331665
1.314114213
279.2361145
1.322446585
268.7993164
1.34148562
259.2027283
1.349915743
241.8540192
1.363333344
226.7354431
1.38415575
213.408844
1.386666298
201.5056763
1.411639214
194.9947357
1.426415801
188.935318
1.428328514
180.6179199
1.441128492
172.8575745
1.453100324
164.9869385
1.464205742
157.740097
1.473819733
151.1829987
1.486423731
143.9502716
1.499343991
138.4791107
1.509965897
132.8890839
1.522242427
129.5950317
1.529255748
126.493248
1.529255748
124.2660522
1.53686142
120.8921432
1.543375134
117.3944702
1.549948096
114.7864304
1.558065772
111.9504318
1.56092155
108.9145203
1.564850807
106.6669846
1.571887255
104.5330276
1.574593782
102.4421844
1.584572434
100.1668015
1.591516852
98.28172302
1.594149351
96.44982147
1.595042825
94.43471527
1.595328212
91.55084229
1.595610261
89.27562714
1.604250789
87.08631134
1.61047101
85.43348694
1.616541862
83.63105011
1.620805621
82.10086823
1.627643347
79.91345978
1.629765868
78.01348877
1.631207824
76.20350647
1.63190341
75.09172821
1.634262919
73.41147614
1.638391137
72.23751831
1.642881751
71.10028076
1.646320224
69.861763
1.648736954
68.40744019
1.655003667
67.13788605
1.662294388
66.03204346
1.667405605
65.08184814
1.670548201
64.04498291
1.671463728
62.38602829
1.673002481
61.32709885
1.673002481
60.30479813
1.673002481
59.41309738
1.673002481
58.54596329
1.673002481
57.799366
1.673613429
56.88968277
1.673613429
55.92052078
1.677541733
54.98633194
1.677541733
The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.
Sulfonation procedure; dried base polymer was added to a 1 L jacketed glass reactor, which was equipped with a Teflon coated agitator. A mixture of concentrated sulfuric acid (98%) and fuming sulfuric acid (20% SO3 in sulfuric acid) was added to the reactor containing base polymer. The reaction was carried out at 90° C. for 24 hours, with constant agitation at 100 RPM.
Work-up; the reaction volume was allowed to cool to room temperature (RT), and was slowly added into a chemical glass beaker with an excess of at least 1 L ice cold DI water. Sulfonated polymer was washed with excess DI water at RT until the supernatant reached a neutral pH. The resulting polymer was then treated with 100 mL 1M NaOH(aq) for 1 hour at RT to convert polymer bound ˜SO3H into ˜SO3Na groups. Polymer was washed again with an excess of DI water at RT until the supernatant reached a neutral pH, then dried in an oven at 100° C. until no further loss on drying was observed. The dried ˜SO3Na functional polymer yield was measured. Reaction compositions for CY15100 and CY15102 are provided in Table X. Table XI displays atomic concentrations for polymers CY15100, CY15102, and CY15087, as measured by XPS. Log differential pore volume plots are presented in
Thrombogenicity was measured by the uPTT assay in which materials were compared to the negative control (plasma alone), positive control (glass beads) and reference beads to determine the degree of contact activation activity. In the uPTT assay, the % change in clot formation over time as compared to the reference materials was determined, then grouped according to: <25% activators, 25-49% moderate activators, 50-74% mild activators, 75-100% minimal and >100% non-activators of the intrinsic coagulation pathway. Polymer CY15100, 82%, was a minimal activator.
TABLE X
Modification Compositions for CY15100 and CY15102
CY15100
CY15102
Base Polymer
CY15045
CY15087
Mass Base Polymer (g)
220.0
80.0
Mass Concentrated Sulfuric Acid (g)
950.0
550.0
Mass Fuming Sulfuric Acid (g)
50.0
30.0
Yield Dry Modified Polymer (g)
355.5
204.6
TABLE XI
Atomic Concentrations (in %) for CY15100,
CY15102, and CY15087
Polymer
Condition
C
N
O
Na
S
CY15100
Bead
65.7
0.2
20.5
8.0
5.7
CY15102
Bead
71.6
0.5
17.2
6.6
4.1
CY15087
Bead
95.5
0.4
4.2
0.0
0.0
CY15087
Ground
98.3
0.2
1.5
0.0
0.0
TABLE XII
Nitrogen Desorption Data for CY15100
Pore Diameter
Average
Cumulative Pore
Range (Å)
Diameter (Å)
Volume (cm3/g)
2072.6-552.5
648.562383
0.000416099
552.5-354.3
410.9223564
0.000905416
354.3-337.5
345.4980521
0.001122701
337.5-311.7
323.5292132
0.001561729
311.7-288.8
299.3515093
0.001919004
288.8-272.0
279.8911375
0.002345465
272.0-252.4
261.4265539
0.002783018
252.4-239.0
245.303922
0.003244227
239.0-225.7
231.9701343
0.004052829
225.7-212.7
218.7962082
0.005280802
212.7-204.5
208.4059706
0.007418375
204.5-131.6
152.2941936
0.087124099
131.6-99.3
110.8101833
0.170472908
99.3-72.3
81.43320551
0.20555251
72.3-62.2
66.46726774
0.212857437
62.2-52.1
56.21812708
0.218554756
52.1-45.7
48.42881742
0.221509707
45.7-39.4
42.03869424
0.223879096
39.4-34.5
36.58507436
0.225521077
34.5-29.1
31.32500867
0.230015257
29.1-25.1
26.83485239
0.230195589
25.1-22.0
23.36857621
0.230286279
22.0-19.4
20.51213112
0.232863812
TABLE XIII
Nitrogen Desorption Data for CY15102
Pore Diameter
Average
Cumulative Pore
Range (Å)
Diameter (Å)
Volume (cm3/g)
1598.8-1238.8
1372.941344
0.026272341
1238.8-946.4
1053.220361
0.092081573
946.4-758.1
831.0482586
0.194131921
758.1-677.1
712.8857859
0.258283006
677.1-529.4
584.7345957
0.38744334
529.4-485.2
505.2928332
0.431560876
485.2-443.2
462.211297
0.481712669
443.2-396.6
417.205311
0.529431372
396.6-361.7
377.5005059
0.571368548
361.7-324.6
341.0804153
0.616836751
324.6-296.1
308.9881056
0.653318693
296.1-271.6
282.7268533
0.684779469
271.6-256.8
263.7792955
0.704908544
256.8-239.4
247.4475287
0.727833901
239.4-230.2
234.5702219
0.739926683
230.2-217.1
223.2037828
0.756372211
217.1-206.8
211.680438
0.769442061
206.8-140.7
160.5985991
0.863794835
140.7-106.0
118.0466801
0.922556622
106.0-82.8
91.24150017
0.968039661
82.8-68.2
73.90381313
1.001829887
68.2-60.9
64.05281388
1.020920023
60.9-52.5
55.98109194
1.044868856
52.5-46.3
48.94597942
1.065247397
46.3-41.2
43.35983259
1.084233305
41.2-37.0
38.81504369
1.106456908
37.0-33.1
34.78421912
1.129603729
33.1-30.0
31.33519542
1.146218801
30.0-27.3
28.4688972
1.162517069
27.3-24.6
25.75215358
1.182048628
24.6-22.4
23.33791231
1.201310022
22.4-19.8
20.89212489
1.229148706
19.8-17.6
18.54892595
1.260822457
TABLE XIV
Mercury Intrusion Data for CY15102
Pore size
Cumulative
Diameter (A)
Intrusion (mL/g)
226247.25
3.02E−30
213156.0625
0.000893837
201297.1875
0.002383566
172619.2656
0.004320214
139526.7344
0.006107889
113150.6484
0.007448644
90544.85156
0.009236319
78737.24219
0.010130156
72447.07031
0.011172966
60339.52344
0.012066803
49074.61719
0.012066803
38783.65625
0.012066803
32031.35742
0.012456137
25154.1582
0.01550037
20919.94336
0.01550037
16226.36035
0.016433783
13231.0293
0.018065026
10569.24219
0.020413134
8346.358398
0.023545867
6777.795898
0.027556093
5545.635742
0.032167129
4347.45166
0.039555997
3496.898926
0.049277436
2839.973145
0.057190847
2592.47998
0.06178461
2267.395264
0.071647309
1831.758789
0.089788206
1510.39563
0.112907536
1394.068237
0.125744253
1294.699707
0.136810422
1207.551147
0.147966579
1132.260498
0.159586608
1065.672974
0.171025708
954.0095215
0.191800222
884.2581177
0.20811981
823.8370972
0.228217274
771.1380615
0.239915013
721.8734131
0.275565475
684.4716797
0.281177133
672.791748
0.283745468
636.3512573
0.295114249
605.4035034
0.309263676
558.758606
0.326112717
518.5050049
0.352752388
483.7310181
0.367008656
453.6919861
0.390547335
426.9628296
0.407471895
403.0959778
0.4232741
382.8546753
0.444355428
362.905426
0.463873088
342.0473328
0.487040371
329.7276001
0.504495382
315.7310791
0.522837102
302.3917236
0.545027971
290.2372131
0.567096949
279.1113586
0.588691056
268.6489563
0.608853817
259.2150879
0.635331511
241.9123993
0.710671127
226.7029877
0.774290979
213.3559113
0.867704988
201.5307922
0.867704988
195.0246887
0.867704988
188.9438019
0.867704988
180.6033783
0.867704988
172.8410034
0.869671643
164.969101
0.869671643
157.8126526
0.87475878
151.1803131
0.905465066
143.936264
0.909094393
138.4554596
0.931292474
132.8584442
0.938616037
129.575531
0.938616037
126.4766693
0.971493781
124.2657852
0.971493781
120.9015427
0.972762465
117.374855
0.977469385
114.7828751
0.981295645
111.9444351
0.981295645
108.8816452
0.981295645
106.6592331
0.986702561
104.5428238
0.996097863
102.4358368
1.000003457
100.1722946
1.003374338
98.26839447
1.006461024
96.44637299
1.008966684
94.41146851
1.012030125
91.54938507
1.015347958
89.25726318
1.018440247
87.0788269
1.021567345
85.42123413
1.024644852
83.62944031
1.028239489
82.1011734
1.02980864
79.91355133
1.032312155
78.00926208
1.034948707
76.20082092
1.037501097
75.09120178
1.039880157
73.4092865
1.042042732
72.23842621
1.043176413
71.09993744
1.047091961
69.86208344
1.047258615
68.40840912
1.049208641
67.1362381
1.05278945
66.0329895
1.05278945
65.08166504
1.053350925
64.04417419
1.054639339
62.38519287
1.055902362
61.32834625
1.060090899
60.30381012
1.062460899
59.41312408
1.063420892
58.54793549
1.064275384
57.79902267
1.066532493
56.88972473
1.068112493
55.92105865
1.072528958
54.9865036
1.072528958
The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.
Sulfonation procedure; dried base polymer was mixed with glacial acetic acid in a 500 mL glass reactor equipped with a Teflon coated mechanical agitator, and heated to 50° C. with agitation set to 100 RPM. A mild sulfonating agent was prepared by adding acetic anhydride (99%) to a 100 mL chemical glass beaker, cooled in an ice bath, and slowly adding concentrated sulfuric acid (98%) over 30 minutes. Temperature of the mixture was monitored and maintained between 15-40° C. by replenishing the ice bath. After completion of the sulfuric acid addition, the reddish-brown viscous liquid was kept at RT for 1 hour, and then slowly added to the reactor. The reaction was allowed to proceed for a specified amount of time.
Work-up; the reaction volume was allowed to cool to room temperature (RT), and was slowly added into a chemical glass beaker with an excess of at least 1 L ice cold DI water. Sulfonated polymer was washed with excess DI water at RT until the supernatant reached a neutral pH. The resulting polymer was then treated with 100 mL 1M NaOH(aq) for 1 hour at RT to convert polymer bound ˜SO3H into ˜SO3Na groups. Polymer was washed again with an excess of DI water at RT until the supernatant reached a neutral pH, then dried in an oven at 100° C. until no further loss on drying was observed. The dried ˜SO3Na functional polymer yield was measured. Reaction compositions for polymers CY14144 and CY15101 are shown in Table XV, below. Atomic concentrations determined by XPS for polymers CY14144, CY12004, CY15101, and CY15087 are presented below, in Table XVI.
Thrombogenicity was measured by the uPTT assay in which materials were compared to the negative control (plasma alone), positive control (glass beads) and reference beads to determine the degree of contact activation activity. In the uPTT assay, the % change in clot formation over time as compared to the reference materials was determined, then grouped according to: <25% activators, 25-49% moderate activators, 50-74% mild activators, 75-100% minimal and >100% non-activators of the intrinsic coagulation pathway. Polymer CY15101, 88%, was a minimal activator.
TABLE XV
Modification Compositions for CY14144 and CY15101
CY14144
CY15101
Base Polymer
CY12004
CY15087
Mass Base Polymer (g)
11.7
80.5
Volume Glacial Acetic Acid (mL)
75
400
Mass Acetic Anhydride (g)
15.5
125.0
Mass Concentrated Sulfuric Acid (g)
10.0
80.0
Reaction Time (hr)
1
2
Yield Dry Modified Polymer (g)
15.2
103.4
TABLE XVI
Atomic Concentrations (in %) for CY14144,
CY12004, CY15101 and CY15087
Polymer
Condition
C
N
O
Na
S
CY14144
Ground
87.0
0.0
8.7
2.5
1.8
CY12004
Bead
88.7
3.4
7.9
0.0
0.0
CY12004
Ground
95.0
0.4
4.7
0.0
0.0
CY15101
Bead
93.3
0.7
5.5
0.4
0.1
CY15087
Bead
95.5
0.4
4.2
0.0
0.0
CY15087
Ground
98.3
0.2
1.5
0.0
0.0
TABLE XVII
Nitrogen Desorption Data for CY14144
Pore Diameter
Average
Cumulative Pore
Range (Å)
Diameter (Å)
Volume (cm3/g)
1316.7-872.8
1006.357045
0.00765904
872.8-760.0
808.4322272
0.01624896
760.0-683.5
717.5974884
0.028034508
683.5-625.7
651.9899544
0.04390322
625.7-580.4
601.3250683
0.063539075
580.4-506.1
537.9818896
0.171853178
506.1-449.5
474.3191955
0.315569993
449.5-395.6
418.9973346
0.494055963
395.6-367.4
380.4061561
0.562885137
367.4-336.7
350.6379611
0.677258819
336.7-297.9
314.7821036
0.775586161
297.9-293.1
295.4222209
0.799779319
293.1-271.2
281.2260787
0.844974101
271.2-254.9
262.5094171
0.885670627
254.9-241.5
247.8251105
0.914384164
241.5-229.9
235.3841146
0.939107638
229.9-218.4
223.8303393
0.963836661
218.4-143.9
165.3652577
1.129471233
143.9-105.5
118.3403917
1.219663568
105.5-85.5
93.18473659
1.269908393
85.5-70.2
76.1345754
1.313809596
70.2-59.9
64.12468772
1.346563035
59.9-51.8
55.16793458
1.375536168
51.8-45.4
48.0976978
1.401122481
45.4-40.2
42.43389408
1.424635177
40.2-36.1
37.88651456
1.449718809
36.1-32.1
33.79019728
1.477483715
32.1-28.9
30.28159181
1.497726602
28.9-26.3
27.45536434
1.516706872
26.3-23.6
24.73777781
1.540207545
23.6-21.1
22.17739744
1.565080566
21.1-19.0
19.93703511
1.59109954
19.0-16.5
17.51596978
1.630846881
TABLE XVIII
Nitrogen Desorption Data for CY15101
Pore Diameter
Average
Cumulative Pore
Range (Å)
Diameter (Å)
Volume (cm3/g)
1633.5-1308.6
1434.817706
0.015869195
1308.6-938.9
1063.085479
0.076011287
938.9-822.1
872.5400938
0.113427572
822.1-664.4
726.153686
0.190492729
664.4-541.2
589.8410779
0.271968628
541.2-495.4
516.2061221
0.306797766
495.4-452.4
471.8824199
0.342922234
452.4-411.9
430.1606805
0.374728484
411.9-373.7
390.8559715
0.406903208
373.7-338.1
354.0440222
0.436879429
338.1-306.8
320.8627294
0.464287882
306.8-282.0
293.2737773
0.487609004
282.0-258.7
269.2384186
0.507679709
258.7-244.7
251.276503
0.519925622
244.7-229.0
236.3042502
0.53405423
229.0-216.9
222.6261956
0.545016489
216.9-207.3
211.8651212
0.55492914
207.3-144.0
163.6239028
0.618758477
144.0-103.4
116.4570854
0.668697015
103.4-85.6
92.59846579
0.694263778
85.6-72.1
77.55016135
0.716487235
72.1-60.7
65.25664927
0.73733967
60.7-53.0
56.18831893
0.752958019
53.0-46.6
49.26725758
0.767344784
46.6-41.5
43.64795708
0.78015016
41.5-37.3
39.09541737
0.794981989
37.3-33.3
35.01993833
0.810085989
33.3-30.2
31.57107231
0.8201347
30.2-27.5
28.71043051
0.829560837
27.5-24.8
25.99331273
0.839975544
24.8-22.6
23.59379328
0.8493402
22.6-20.0
21.1163566
0.861908143
20.0-17.8
18.73336683
0.874402144
TABLE XIX
Mercury Intrusion Data for CY15101
Pore size
Cumulative
Diameter (A)
Intrusion (mL/g)
226247.25
3.367E−30
213156.0625
0.001661795
201297.1875
0.002658872
172619.2656
0.005317744
139526.7344
0.007976616
113150.6484
0.009638411
90544.85156
0.012297283
78737.24219
0.014125257
72447.07031
0.015620873
60339.52344
0.017947385
49556.56641
0.01949878
38738.37109
0.021929506
31002.00586
0.023903539
25333.7832
0.02616792
20724.62109
0.029177248
16168.99121
0.033409968
13230.375
0.037765641
10563.43555
0.044586275
8346.731445
0.054462213
6776.340332
0.0666546
5536.147949
0.083998173
4342.036621
0.107802272
3501.501953
0.137485042
2837.420654
0.177576199
2594.672363
0.200087309
2269.617432
0.233489379
1831.204224
0.295208424
1510.503906
0.360582143
1395.643555
0.392902017
1293.973755
0.421268374
1207.494141
0.447410613
1131.894531
0.47241658
1065.193237
0.49649471
953.9039307
0.535679519
884.3017578
0.568524599
823.786377
0.597846568
771.5706177
0.616960466
722.1925049
0.691450536
684.2458496
0.697761118
672.2320557
0.703246117
636.7992554
0.713504672
604.4926758
0.726847529
558.8725586
0.746505737
517.9966431
0.774387836
483.9524536
0.799027622
453.7037354
0.824069798
426.9303894
0.846621335
403.1401672
0.898474514
382.6773987
0.91877532
362.9386292
0.946397841
342.2199707
0.946397841
330.153656
0.953894079
315.6123962
0.954481184
302.6812439
0.954481184
290.4436646
0.967425823
279.009491
0.974567354
268.8323975
0.974567354
259.2565308
0.974567354
241.9353333
1.028741002
226.8330078
1.048289418
213.444046
1.065926313
201.5080414
1.074228048
195.0001221
1.095143437
188.9437103
1.106776357
180.6530914
1.11556828
172.9412994
1.127364159
164.9789429
1.139592171
157.7405396
1.150992036
151.1612091
1.161784291
143.9489746
1.172875285
138.4779053
1.185242534
132.8603821
1.194525123
129.5736542
1.200321555
126.4793472
1.207967401
124.2483292
1.213427901
120.9080048
1.221876502
117.3827286
1.223723292
114.771225
1.23161757
111.937149
1.237899184
108.9081039
1.239180923
106.6535568
1.245096564
104.5474396
1.24916625
102.455368
1.25267899
100.1680145
1.261325955
98.2784729
1.261325955
96.45231628
1.267885208
94.40316772
1.274962544
91.53180695
1.279593945
89.26702118
1.285915971
87.08314514
1.285915971
85.42582703
1.287682652
83.6335144
1.29400897
82.10058594
1.300267935
79.91345978
1.30387032
78.01080322
1.308264375
76.19985962
1.313777924
75.09228516
1.318249345
73.41210175
1.321508646
72.23653412
1.323805094
71.09803772
1.32500124
69.86273193
1.334167719
68.40810394
1.336985707
67.13769531
1.340026617
66.03487396
1.340026617
65.0819931
1.340224981
64.04338074
1.340224981
62.38589478
1.346690297
61.32817841
1.358168244
60.30670166
1.358168244
59.41316605
1.358532906
58.54763031
1.358532906
57.79816818
1.358532906
56.88824844
1.358532906
55.92269516
1.366921306
54.98662186
1.373521209
The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.
Among various “mild” sulfonating agents acetyl sulfate (prepared from 98% conc. Sulfuric acid and acetic anhydride at low temperatures) is known to be very efficient for DVB or styrene based polymeric materials. Sulfonation is usually done at 50° C. for several hours using equimolar amounts of acetyl sulfate and DVB or styrene based polymers. Sulfonation occurs mainly at benzene ring and unreacted double bonds (in DVB based cross-linked polymeric porous beads) could be preserved for further functionalization. Usually after sulfonation with acetyl sulfate, the polymer is converted into ˜SO3Na form and can be graft copolymerized with N-vinyl pyrrolidone (in bulk with benzoyl peroxide as initiator) or in water solutions (using sodium persulfate initiator). Resulting sulfonated polymer is “coated” with poly(N-vinylpyrrolidone) to make hemocompatible material capable of removing K+ cations from physiological fluids.
The base polymer selected for this modification was polymer CY15044. The sulfonation and workup were carried out as described in Example 4, using 45.0 g dry CY15044 polymer, 150 mL glacial acetic acid, 62.0 g acetic anhydride, and 40.0 g concentrated sulfuric acid. The resulting sulfonated polymer, in ˜SO3Na form, was rewet in DI water in a 1 L jacketed reaction vessel fitted with a Teflon coated agitator. DI water was removed from the vessel, and a solution composed of 75 mL NVP monomer, 1.7 g sodium persulfate, and 25 mL DI water was added. The reaction was allowed to proceed for 72 hours at 70° C. with agitation speed set to 100 RPM. Resulting poly(NVP) coated polymer was washed five times using 200 mL DI water, and dried in a vacuum oven until no further loss on drying was observed. Cumulative pore volume data for polymer CY15048 is shown below, in Table XX. A log differential pore volume plot is shown in
Thrombogenicity was measured by the uPTT assay in which materials were compared to the negative control (plasma alone), positive control (glass beads) and reference beads to determine the degree of contact activation activity. In the uPTT assay, the % change in clot formation over time as compared to the reference materials was determined, then grouped according to: <25% activators, 25-49% moderate activators, 50-74% mild activators, 75-100% minimal and >100% non-activators of the intrinsic coagulation pathway. Polymer CY15048, 94%, was a minimal activator.
TABLE XX
Nitrogen Desorption Data for CY15048
Pore Diameter
Average
Cumulative Pore
Range (Å)
Diameter (Å)
Volume (cm3/g)
4355.2-828.4
944.3942734
0.0001057
828.4-474.0
558.6159743
0.000283797
474.0-303.3
351.2170892
0.000357942
303.3-224.3
251.4508466
0.000719278
224.3-216.6
220.2880162
0.001144201
216.6-208.1
212.161881
0.001932015
208.1-143.6
163.4823625
0.075370749
143.6-108.1
120.3913517
0.239861146
108.1-81.7
90.85045572
0.292261423
81.7-71.8
76.00202402
0.305608258
71.8-60.2
64.83123041
0.320783836
60.2-52.6
55.8248996
0.329250736
52.6-46.6
49.1740518
0.335209946
46.6-41.3
43.5462226
0.339923553
41.3-37.4
39.07691383
0.349323983
37.4-32.5
34.52204235
0.351977397
32.5-29.4
30.76412188
0.352966945
29.4-27.3
28.24001433
0.353787455
27.3-24.6
25.76447932
0.355166927
24.6-22.3
23.26898468
0.357207636
22.3-19.9
20.87422635
0.360962494
19.9-17.5
18.46778237
0.367188172
The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.
The base polymer selected for this modification was polymer CY15042. The sulfonation and workup were carried out as described in Example 4, using 45.0 g dry CY15042 polymer, 200 mL glacial acetic acid, 62.0 g acetic anhydride, and 40.0 g concentrated sulfuric acid. The reaction was allowed to proceed for 2 hours. The resulting dried sulfonated polymer, in ˜SO3Na form, was added to a 1 L jacketed reaction vessel fitted with a Teflon coated agitator. 140.0 g N-vinylpyrrolidone monomer and 2.0 g benzoyl peroxide were added to the reactor. The reaction was allowed to proceed for 24 hours at 70° C. with agitation speed set to 100 RPM. Resulting poly(N-vinylpyrrolidone) coated polymer was washed five times using 200 mL DI water, and dried in a vacuum oven until no further loss on drying was observed. Table XXI, below, displays cumulative pore volume data for polymer CY15049.
TABLE XXI
Nitrogen Desorption Data for CY15049
Pore Diameter
Average
Cumulative Pore
Range (Å)
Diameter (Å)
Volume (cm3/g)
6798.1-997.4
1113.294549
0.002499046
997.4-529.0
628.6356118
0.005782394
529.0-503.0
515.3445059
0.00652485
503.0-431.3
461.4274588
0.007796961
431.3-320.8
359.3702778
0.010896953
320.8-317.4
319.0643487
0.011833304
317.4-274.0
292.3669097
0.013396248
274.0-230.2
248.1049882
0.016483381
230.2-225.4
227.7447013
0.017366617
225.4-211.6
218.0383103
0.018833905
211.6-195.5
202.8978228
0.029306436
195.5-143.0
160.6741284
0.494786051
143.0-99.0
112.5005572
0.779812896
99.0-82.5
89.05063735
0.848450234
82.5-69.8
74.92200629
0.902458565
69.8-59.0
63.37885992
0.947842682
59.0-51.2
54.4553105
0.981969695
51.2-44.8
47.47101253
1.011973922
44.8-39.4
41.69146063
1.039279282
39.4-35.3
37.10279099
1.066468142
35.3-31.3
33.03019211
1.096075821
31.3-28.2
29.57468036
1.118921801
28.2-25.5
26.70738162
1.143080339
25.5-22.8
23.944141
1.17115692
22.8-20.4
21.43590284
1.201324419
20.4-18.2
19.13416412
1.23163662
The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.
Units of human pRBC were allowed to equilibrate to room temperature for 30 minutes, where the units were gently mixed for 15 minutes. A blood spike was inserted into the unit and samples for the initial hemoglobin (Hb) and potassium concentrations were taken. The blood spike line was attached to the top port of the polymer containing filtration device, and a sample collection line attached to the bottom port. A pinch clamp was fitted on the sample collection line for flow control. Approximately one bed volume, 30 mL, was flushed through the device into a waste container to purge the device of normal saline solution. The sample collection tube was placed over 15 mL conical tubes where 12 mL fractions of pRBCs were collected at a flow rate of about 3-3.5 mL/min until the unit was completely filtered. Sample tubes were centrifuged for 15 minutes at 4600 RPM at 4° C. Plasma supernatant from each sample tube was collected and the plasma free hemoglobin level was determined by an absorbance read at 450 nm and potassium levels were measured with a potassium ion-selective electrode. The percentage of initial free hemoglobin removed during single-pass filtration, averaged from three trials, is presented in
The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.
Polymer CY14144 has been tested in a dynamic competitive system evaluating albumin (30 mg/mL) and myoglobin (100 mg/L) removal from a PBS solution with 8 mEq/L potassium. This model has been designed to reflect clinical albumin and myoglobin (rhabdomyolysis) values. The dynamic system allows for the continuous measurement of protein adsorption by the polymer beads at two UV wavelengths. As long as the surrogate proteins, such as albumin and myoglobin, have different UV absorption profiles, the two protein surrogates can be measured simultaneously, providing competitive adsorption conditions. This allows a rapid assessment of polymer performance for the simultaneous adsorption of target and non-target factors under flow conditions; a key parameter to assess studies that balance sorption with hemocompatibility. The dynamic system has been fully calibrated (absorbance and flow conditions) and was used to measure binding with a 6 mL polymer filled device at a flow rate of 6 mL/min for five hours at room temperature. CY14144 has a robust myoglobin adsorption, potassium removal and demonstrated good selectivity with minimal albumin removal. Dynamic recirculation data for CY14144, averaged from 7 trials, is shown below in
Thrombogenicity was measured by the uPTT assay in which materials were compared to the negative control (plasma alone), positive control (glass beads) and reference beads to determine the degree of contact activation activity. In the uPTT assay, the % change in clot formation over time as compared to the reference materials was determined, then grouped according to: <25% activators, 25-49% moderate activators, 50-74% mild activators, 75-100% minimal and >100% non-activators of the intrinsic coagulation pathway. Shown below, in Table XXII, is a comparison of thrombogenicity for two different potassium removing polymers. Polymer CY14144 exhibits minimal thrombogenic activity while still removing potassium and myoglobin simultaneously in a dynamic recirculation model in phosphate buffered saline (PBS). In comparison, potassium sorbent CY14022 is a moderate activator of the intrinsic coagulation pathway by the uPTT assay and is ineffective in myoglobin removal.
TABLE XXII
Myoglobin and Potassium Removal from PBS in a
Dynamic Recirculation Model
Myoglobin
Potassium
Polymer
uPTT
Removal
Removal
CY14144
87%
71.63%
25.3%
CY14022
59%
5.94%
66.07%
The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.
Additionally, polymer CY14144 is able to remove significant levels of potassium from blood plasma in a dynamic recirculation model. The normal range for blood potassium is 3.5-5 mEq/L while a patient suffering from hyperkalemia might have blood potassium levels up to 7-7.5 mEq/L. Reperfusion of plasma with a starting concentration of potassium 7.45 mEq/L through a device filled with polymer CY14144 under recirculation conditions that mimic the clinical application reduced the potassium concentration to 4.52 mEq/L (a 2.93 mEq/L reduction) in 5 hours.
The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.
Young, Wei-Tai, Gruda, Maryann, Capponi, Vincent, Guliashvili, Tamaz, Golobish, Thomas, O'Sullivan, Pamela, Scheirer, Andrew, Dan, Vi, Chan, Phillip
| Patent | Priority | Assignee | Title |
| 11723916, | Oct 22 2015 | CYTOSORBENTS CORPORATION | Multi-functional hemocompatible porous polymer bead sorbent for removing protein based toxins and potassium from biological fluids |
| Patent | Priority | Assignee | Title |
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